Compositions and Methods for the Transfer of a Hexosamine to a Modified Nucleotide in a Nucleic Acid

ABSTRACT

Nucleic acids comprising β-glucosaminyloxy-5-methylcytosine; compositions, kits and methods of producing the nucleic acids using a glycosyltransferase; and methods of using the nucleic acids are described.

REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of each of the following patentapplications, the entire disclosures of each of which are herebyincorporated by reference into the present application: U.S. 61/611,295,filed Mar. 15, 2012; U.S. Application No. 61/722,968, filed Nov. 6,2012; U.S. Application No. 61/723,427, filed Nov. 7, 2012; and U.S.Application No. 61/724,041, filed Nov. 8, 2012. Also incorporated byreference in their entireties are the following applications filed onthe same day as the present application: Attorney Docket No. NEB-364-US,“Compositions and Methods for Oxygenation of Nucleic Acids Containing5-Methylpyrimidine”; Attorney Docket No. NEB-363-US, “Mapping CytosineModifications”; and Attorney Docket No. NEB-354-US, “Methods andCompositions for Discrimination Between Cytosine and ModificationsThereof, and for Methylome Analysis.”

BACKGROUND

Modified nucleotides in the genome are associated with epigenetics ingeneral and regulation of transcriptional activation in particular.Detection and/or mapping of modified nucleotides in the genome areimportant for the understanding of the relationship of phenotype togenotype. Examples of modified nucleotides are 5-hydroxymethylcytosine(5-hmC) and 5-methylated cytosine (5-mC). One approach todetection/mapping of these modified nucleotides is bisulfite sequencing.However, this technique cannot differentiate between the two forms. Analternative approach has been to use T4-β-glucosyltransferase (BGT) totransfer glucose from uridine diphospho-glucose (UDP-Glc) to 5-hmC. Theglucose is amenable to chemical derivatization with an azide or thiogroup (US Published Application No: 2011/0236894 and InternationalPublished Application No: WO 2011/02581). Alternative substrates for BGTand other glycosyltransferases would prove useful for enhancing thesensitivity and specificity for 5-hmC and other modified nucleotidesanalysis as well as for multiplex analysis.

SUMMARY OF THE INVENTION

The present invention is derived, in part, from the discovery that aβ-glycosyltransferase can be used to transfer glucosamine to modifiednucleotides such as 5-hmC. As far as the inventors are aware,glucosaminated nucleic acids do not exist in nature and their synthesishas now been made possible for the first time using the enzymaticmethods described in this application. The ability to introducesite-specific modifications to a nucleic acid permits site-specificlabeling of a nucleic acid, such as through the amino group ofglucosamine. In conjunction with a restriction enzyme that distinguishesglucosaminated and non-glucosaminated nucleic acids, site-specificmodification permits selective control over cleavage of the nucleicacid. Moreover, the methods of the invention can be used to specificallymodify 5-hmC residues in a nucleic acid, facilitating their subsequentcharacterization.

Accordingly, in one aspect, the invention provides a composition (suchas an enzyme preparation) or a kit useful for modifying a nucleic acid.The composition or kit includes UDP-glucosamine (UDP-GlcN) and aβ-glycosyltransferase, such as BGT. Wild-type and mutant forms of BGTare each useful, as described in Example 1. Thus, if BGT variants areused, the variant may be a fragment retaining enzymatic activity, or maycontain amino acid substitutions varying the amino acid sequence, whileoptionally retaining at least 95% identity, at least 90% identity, atleast 85% identity, at least 80% identity, at least 75% identity, or atleast 70% identity with wild-type BGT or an active fragment thereof. Theglucosamine is optionally a 2-glucosamine or a 6-glucosamine, and caninclude a label, such as a radioactive label. The composition or kitalso typically includes a buffer having a pH between 6 and 8, such asbetween 6.8 and 7.4, for example. Other components, such as a label oran antibody or other binding moiety with an affinity for glucosamine mayalso be included. In some embodiments, the composition or kit mayinclude a restriction enzyme capable of cleaving a nucleic acid at asite comprising β-glucosyl-5-hydroxymethylcytosine (β-5 gmC) but not asite comprising β-2-glucosaminyl-5-hydroxymethylcytosine.

The invention also provides methods for labeling a modified nucleotide(such as 5-hmC) in a nucleic acid. The methods include combining thenucleic acid with a composition containing both the UDP-glucosamine andthe β-glycosyl transferase under conditions permitting the glucosamineto become covalently attached to the modified nucleotide in the nucleicacid. These conditions generally involve appropriate pH (optionallyfacilitated by a buffer) and temperature. In some embodiments, themethods further include subsequently reacting the covalently attachedglucosamine with a label, such as a radioactive label, a fluorophore, adye, or an affinity label (such as biotin). Some methods of theinvention include enriching, detecting, isolating and/or identifying theposition of the glucosamine-attached nucleotide in the nucleic acid.

For example, the invention provide methods for detecting a modifiednucleotide in a genome or genomic fragment by combining a nucleic acidhaving a modified nucleotide with UDP-glucosamine and BGT; permittingthe glucosamine to become covalently attached to the modified nucleotidein the nucleic acid; and detecting the modified nucleotide either bylabeling the glucosaminated modified nucleotide or bymodification-specific enzymatic cleavage using, for example, an enzymecapable of cleaving 5-hmC and 5-gmC, but not glucosamine-containing5-hmC or labeled glucosamine-containing 5-hmC sites. A genomic fragmentis typically at least 100 bases in length, and may be at least 1000bases; at least 10,000 bases; at least 100,000 bases; at least 1,000,000bases; or greater in length.

The invention also provides methods for detecting a 5-hmC in a nucleicacid or nucleic acid fragment with a composition containing both theUDP-glucosamine and the β-glycosyltransferase by converting amethylcytosine residue into a hydroxymethylcytosine by means of achemical oxidizing agent or a cytosine oxygenase enzyme and thenpermitting the glucosamine to become covalently attached to the oxidizednucleotide.

The invention also provides methods for detecting a 5-hmC in a nucleicacid or nucleic acid fragment with a composition containing both theUDP-glucosamine and the β-glycosyltransferase by converting aformylcytosine or carboxylcytosine residue into a hydroxymethylcytosineby means of a chemical reducing agent, such as sodium borohydride, andthen permitting the glucosamine to become covalently attached to thereduced nucleotide.

In another aspect, the invention provides nucleic acids modified toinclude β-5 gmC residues in which one or more of the hydroxyl groups atthe 2- and/or 6-positions of the glucosyl moiety have been replaced withamino groups. The modified β-5 gmC is typically aβ-glucosaminyloxy-5-methylcytosine, such asβ-2-glucosaminyloxy-5-methylcytosine orβ-6-glucosaminyloxy-5-methylcytosine. The nucleic acid may be labeled,such as with a fluorophore, an affinity label, a radioactive label or adye. In some embodiments, the nucleic acid is a mammalian genome orgenome fragment.

The invention also provides methods for labeling such a nucleic acid byreacting it with a label (e.g., a fluorophore, an affinity label, aradioactive label or a dye) at a modified (e.g., aβ-glucosaminyloxy-5-methylcytosine). Similarly, the invention providesmethods for detecting a modified nucleotide in a nucleic acid, such as agenome or genomic fragment, by labeling the modified nucleotide asdescribed above, and subsequently detecting the presence or absence ofthe label, where the presence of the label is indicative of the presenceof the modified nucleotide.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows how glucosamines naturally occur in several aminoglycosideantibiotics (e.g., Kanamycin, Paromomycin, Amikacin, etc.) isolated fromStreptomyces strains.

FIG. 2 shows (A) a detection scheme for 5-hmC in DNA usingfunctionalized UDP-Glc derivatives and (B) chemical structures ofsynthetic UDP-Glc derivatives whose syntheses are described in Example2.

FIG. 3 shows a synthetic scheme for preparing UDP-6-glucosamine(UDP-6-GlcN) 3 (A) and UDP-2-glucosamine (UDP-2-GlcN) 4 (B).

FIG. 4 shows images of agarose gels depicting the results of enzymatictransfer experiments with wild-type BGT (top row), BGT/Y261L (middlerow), and wild-type AGT (bottom row) using UDP-Glc derivatives assubstrates. (A) natural UDP-Glc, (B) UDP-6-Azido-Glc 1, (C) UDP-6-GlcN3, and (D) UDP-2-GlcN 4.

FIG. 5 shows images of agarose gels depicting the results of enzymatictransfer experiments with wild-type BGT (top row), BGT/Y261L (middlerow), and wild-type AGT (bottom row) using UDP-Glc derivatives assubstrates. UDP-2-Azido-Glc 2 (A), UDP-2-Keto-Glc 5 (B), UDP-GlcUA (C),and UDP-GlcNAc (D).

FIG. 6 shows images of agarose gels depicting the results ofUDP-2-Azido-Glc 2 inhibitory tests with wild-type BGT (top row) andBGT/Y261L (bottom row).

FIG. 7 shows a schematic of an assay for identifying hmC in DNA.

FIG. 8 shows images agarose gels depicting the results of labeling testsof (A) 6-Azido-Glc- and (B) 6-glucosamine-containing syntheticoligonucleotides.

FIG. 9 shows an image of an agarose gel depicting the results ofrestriction endonuclease cleavage of 5-hmC- and β-5 gmC-containingoligonucleotides.

FIG. 10 shows images of agarose gels depicting the results of labelingof a 6-Azido-Glc-modified oligonucleotide at different temperatures.

FIG. 11 shows images of agarose gels depicting the results of labelingof 6-glucosamine- and 2-glucosamine-modified oligonucleotides.

FIG. 12 shows images of agarose gels depicting the results of labelingof 6-glucosamine-modified oligonucleotides at different pHs.

FIG. 13 shows images of agarose gels depicting the results of labelingof 6-glucosamine-modified oligonucleotides at different temperatures.

FIG. 14 shows an image depicting the results of an anti-D-glucosaminebinding assay.

DETAILED DESCRIPTION OF EMBODIMENTS

Certain hexosamines may be used to place a reactive group on a targetmodified nucleotide in a nucleic acid for the purposes of reacting witha label for sequencing and/or detection. For example, a hydroxyl group,a formyl group or a carbonyl group on a methylated cytosine can bereplaced with a GlcN. One of the advantages of using GlcN over glucosecofactors modified with non-naturally occurring groups (e.g.,azido-modified sugars) is the availability of various GlcN-modifyingenzymes (e.g., Hexokinase, Sigma-Aldrich, St. Louis, Mo.) andGlcN-specific antibodies (e.g., anti-D-GlcN antibody, Abcam PLC,Cambridge, Mass.) that could react or interact with the amine on theglucosaminated cytosine. Fortuitously, the transfer of GlcN to an hmChas been achieved using glucosyltransferases or mutantglucosyltransferases while these enzymes cannot efficiently react withUDP-GlcNAc or UDP-GlcUA.

Exemplary glucosyltransferases are found in bacteriophage, such as T4.The T4 glucosyltransferases show little DNA sequence specificity,suggesting a mechanism of non-specific DNA binding combined withspecific 5-hmC recognition.

Variants of the T4 glucosyltransferases can be used. For example, thestructure of T4 BGT and the identities of key residues in the enzyme arewell understood, facilitating the construction of forms of the proteinincorporating one or more amino acid deletions or substitutions. T4 BGTis a monomer comprising 351 amino acid residues and belongs to theα/βprotein class. It is composed of two non-identical domains, bothsimilar in topology to Rossmann nucleotide-binding folds, separated by adeep central cleft which forms the UDP-Glc binding site. Amino acidsparticipating in the interaction with UDP include Ile238 (interactionswith N3 and O4 of the base); Glu272 (interactions with O2′ and O3′ ofthe ribose); Ser189 (interacting with O11 of the α-phosphate); Arg191(interacting with O12 of the α-phosphate); Arg269 (interacting with 06of the α-phosphate and O22 of the β-phosphate); and Arg195 (interactingwith O21 and O22 of the β-phosphate). Glu22 and Asp100 have beenproposed to participate in the catalytic mechanism and other residueshave been proposed to be involved in DNA binding or interactions withthe UDP-associated sugar (Moréra et al. (1999) “T4 phagebeta-glucosyltransferase: substrate binding and proposed catalyticmechanism.” J. Mol. Biol. 292(3):717-730, the entire disclosure of whichis incorporated herein by reference).

Accordingly, a variant glucosyltransferases can be used to add a sugarto a nucleic acid. Variants optionally include an amino acid sequence atleast 70% (e.g. at least 75%, at least 80%, at least 82%, at least 84%,at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100%) identical to amino acids 1-351, 10-272 or 22-272 of T4 BGT. Asassays for glycosylated nucleic acids (e.g. changes in susceptibility tocleavage by a glycosylation-sensitive endonuclease) are readilyavailable, screening for variants retaining enzymatic activity isrelatively straightforward.

2-GlcN is an amino sugar and a prominent precursor in the biochemicalsynthesis of glycosylated proteins and lipids. 2-GlcN is part of thestructure of the polysaccharides chitosan and chitin, which compose theexoskeletons of crustaceans and other arthropods, cell walls in fungiand many higher organisms. 2-GlcN is one of the most abundantmonosaccharides. 2-, 3-, 4-, and 6-GlcNs are present in severalaminoglycoside antibiotics (e.g., Kanamycin, Paromomycin, Amikacin,etc.) isolated from Streptomyces strains (FIG. 1). UDP-GlcN was observedin E. coli cells depleted of acetyltransferase activity of GlmU. GlmUcatalyzes the last two sequential reactions in the de novo biosyntheticpathway for UDP-GlcNAc, and is responsible for the acetylation ofGlcN-1-phosphate (GlcN-1-P) to give GlcNAc-1-P, and for the uridyltransfer from UTP to GlcNAc-1-P which produces UDP-GlcNAc [Pompeo F,Bourne Y, van Heijenoort J, Fassy F, Mengin-Lecreulx D (2001) Dissectionof the bifunctional Escherichia coli N-acetylglucosamine-1-phosphateuridyltransferase enzyme into autonomously functional domains andevidence that trimerization is absolutely required forglucosamine-1-phosphate acetyltransferase activity and cell growth. JBiol Chem. 276(6), 3833-3839].

Contacting glucosamine-containing 5-hmC with a restriction enzyme orpolymerase can be envisioned for sequencing purposes. Nucleic acidscontaining modified bases that have been GlcN-modified may be sequenceddirectly in single-molecule real-time (SMRT) DNA sequencing forhigh-throughput, base-resolution 5-hmC detection using a sequencerprovided, for example, by Pacific Biosciences, Menlo Park, Calif. InSMRT DNA sequencing, polymerase kinetic signatures provide the basis fordiscrimination between the DNA modifications. The polymerase rate at andaround the modified base position in the DNA template is slowed comparedto unmodified DNA, and this retardation is enhanced when 5-hmC isreacted with GlcN to provide a unique kinetic signature in SMRT DNAsequencing. This signal is more easily detectable than the signal fromglycosylated 5-hmC or from 5-hmC and information from this enhancedsignal can be used algorithmically to increase the confidence of 5-hmCassignments.

Direct detection of 5-hmC in DNA samples has been achieved by nanoporeamperometry [Wanunu M, Cohen-Karni D, Johnson R R, Fields L, Benner J,Peterman N, Zheng Y, Klein M L, Drndic M. (2011) Discrimination ofmethylcytosine from hydroxymethylcytosine in DNA molecules. J Am Chem.Soc. 133(3), 486-492. In this technique, ion current signatures for DNAmolecules threaded through nanopores are associated with the polarity ofthe cytosine modification. Nucleic acids containing modified bases thathave been GlcN-modified may be sequenced directly in nanoporeamperometry DNA sequencing for high-throughput, base-resolution 5-hmCdetection. Because GlcNs are charged in buffers at and aroundphysiological pH, the modification of 5-hmC with GlcN can provide aunique ion current signature in nanopore amperometry DNA sequencing andthis information can be used to increase the confidence of 5-hmCassignments.

Glucosamine-containing 5-hmC residues can be detected using GlcNspecific enzymes. For example, GlcN can be phosphorylated by an enzymehexokinase (HK, EC 2.7.1.1.) and adenosine-5′-triphosphate (ATP) to formGlcN-6-phosphate and adenosine-5′-diphosphate (ADP). Commerciallyavailable detection kits for GlcN assay (K-GAMINE; MegazymeInternational Ireland, Bray, Co. Wicklow, Ireland) consist of enzymaticsequential reactions (hexokinase, glucose-6-phosphate dehydrogenase,phosphoglucose isomerase, and GlcN-6-P deaminase), in which the amountof nicotinamide adenine dinucleotide phosphate (NADPH) formed isstoichiometric with the amount of GlcN. The NADPH is measured by theincrease in absorbance at 340 nm. Such kits are suitable for manual,auto-analyser and microplate formats. Once GlcN-containing 5-hmC isconverted to GlcN-6-phosphate, various enzymatic-based assays candesigned for the detection of 5-hmC, including the use ofGlcN-6-phosphate deaminase (EC 3.5.99.6), GlcN-6-phosphate isomerase (EC3.5.99.6), GlcN-6-phosphate N-acetyltransferase (EC 2.3.1.4), or otherenzymes known in the art.

Another example of enzymes that can modify GlcN residues areHeparan-sulfate 6-O-sulfotransferases (HS6ST) (EC 2.8.2.-),UDP-GlcNAc6-O-sulfotransferase (EC=2.8.2.-), and related enzymes knownin the art. HS6ST is a 6-O-sulfation enzyme which catalyzes the transferof sulfate from 3′-phosphoadenosine 5′-phosphosulfate (PAPS) to position6 of the N-sulfoglucosamine residue (GlcNS) of heparan sulfate.O-sulfotransferase enzymatic-based assays can used for the detection of5-hmC by or after the conversion of GlcN-containing 5-hmC intoGlcN-6-sulfate.

Glucosamine-containing 5-hmC can be detected using radiolabeledUDP-[3H]GlcN, UDP-[14C]GlcN, UDP-[3H,14C]GlcN, [α-32P]UDP-GlcN or[β-32P]UDP-GlcN, and BGT to generate a radioactive output and/orradioactively labeled GlcN-containing nucleotides which can besubsequently detected directly by scintillation counting.

Glucosamine-containing 5-hmC can provide a target for pull-down byimmobilized J-binding protein 1. J-binding proteins from Africantrypanosomes and related kinetoplastids which specifically bind to DNAcontaining the J-base (β-glucosyl-5-hydroxymethyluracil) have been shownto cross-react with β-5 gmC containing DNA [Robertson A B, Dahl J A,Vågbø C B, Tripathi P, Krokan H E, Klungland A (2011) A novel method forthe efficient and selective identification of 5-hmC in genomic DNANucleic Acids Res. 39(8), e55]. Commercially available enrichment kitsfor the specific enrichment of 5-hmC containing DNA (Quest 5-hmC™ DNAEnrichment Kit; Zymo Research, Irvine, Calif.) featuring J-base bindingprotein (JBP) consist of converting 5-hmC to 5 gmC using aglycosyltransferase (e.g., BGT), follow by adding JBP immobilized inmagnetic beads, and then washing and eluting the enriched 5-hmCcontaining DNA. It is conceivable that JBP proteins may also bind toother DNA glycosylated forms, including glucosamine-containing 5-hmC.

Glucosamine-containing 5-hmC can be chemically oxidized with sodiumperiodate, which converts the sugar vicinal hydroxyl groups toaldehydes, and further modified with an aldehyde-reactive probe. Analdehyde-reactive probe can be, for instance, a biotin hydrazide (e.g.,Z-Link Hydrazide-Biotin Reagents, Thermo Scientific, Waltham, Mass.) forpull-down and affinity-purification using streptavidin or avidin beads;a fluorophore hydroxylamine (e.g., Alexa Fluor® 488 Hydroxylamine, LifeTechnologies, Carlsbad, Calif.) or a fluorophore hydrazide (e.g., AlexaFluor® 647 Hydrazide, Life Technologies, Carlsbad, Calif.) forfluorescence-based detection.

Glucosamine-containing 5-hmC residues can be detected using GlcNspecific chemistry. Specific incorporation of GlcN into 5-hmC residuesvia UDP-GlcN transglycosylation reaction of mediated by BGT permits thelabeling of some or all available 5-hmC bases in the acceptor DNA at theGlcN amino functional group. Amino group reactions offer simplicity andversatility to the identification of 5-hmC in DNA samples by means ofreadily available and inexpensive amino-reactive fluorophores oraffinity reagents containing a label L. In one embodiment, the label Lcan be used for 5-hmC enrichment using an affinity label such as biotinor other binding ligand that can be installed onto GlcN-containing 5-hmCresidues via activated ester chemical coupling. In another embodiment,GlcN-containing 5-hmC residues can be directly labeled with detectableprobe, such as amine-reactive fluorescent or fluorogenic reagents.

The label L of the substrate can be chosen by those skilled in the artdependent on the application for which the fusion protein is intended.Labels are such that the labeled GlcN-containing nucleotide carryinglabel L is easily detected or separated from its environment. Otherlabels considered are those which are capable of sensing and inducingchanges in the environment of the labeled GlcN-containing nucleotide orlabels which aid in manipulating the GlcN-containing nucleotide by thephysical and/or chemical properties of the GlcN substrate specificallyintroduced into the nucleotide.

The label L is designed to covalently react with the amino group ofGlcN-containing nucleotides. Examples of amino-reactive groups areisocyanates, isothiocyanates, active esters (e.g., succinimidyl esters,sulfosuccinimidyl esters, tetrafluorophenyl esters,sulfotetrafluorophenyl esters, sulfodicholorphenol esters), carboxylicacids, acid halides, anhydrides, acyl azides, dichlorotriazines, andsulfonyl chlorides, which form ureas, thioureas, carboxamides, orsulfonamides upon reaction with amines. Other amine-reactive groups arealdehydes, dialdehydes, ketones, vinyl sulfones, vinyl esters, alkylhalides, peroxides, and epoxides.

A label L as understood in the context of the invention is a substituentdifferent from hydrogen or from standard functional groups, inparticular different from hydrogen, hydroxy, amino, halogen,carboxylate, carboxamide, carboxylic ester, nitrile, cyanate,isocyanate, sulfonate, sulfonamide, sulfonic ester, aldehyde, ketone,ether, and thioether substituent. Examples of a label include aspectroscopic probe such as a fluorophore or a chromophore, a magneticprobe or a contrast reagent; a radioactively labeled molecule; amolecule which is one part of a specific binding pair which is capableof specifically binding to a partner; a molecule that is suspected tointeract with other biomolecules; a library of molecules that aresuspected to interact with other biomolecules; a molecule which iscapable of crosslinking to other molecules; a molecule which is capableof generating hydroxyl radicals upon exposure to H₂O₂ and ascorbate,such as a tethered metal-chelate; a molecule which is capable ofgenerating reactive radicals upon irradiation with light, such asmalachite green; a molecule covalently attached to a solid support,where the support may be a glass slide, a microtiter plate or anypolymer known to those proficient in the art; a nucleic acid or aderivative thereof capable of undergoing base-pairing with itscomplementary strand; a lipid or other hydrophobic molecule withmembrane-inserting properties; a biomolecule with desirable enzymatic,chemical or physical properties; or a molecule possessing a combinationof any of the properties listed above.

Further labels L are positively charged linear or branched polymerswhich are known to facilitate the transfer of attached molecules overthe plasma membrane of living cells. This is of particular importancefor substances which otherwise have a low cell membrane permeability orare in effect impermeable for the cell membrane of living cells. Anon-cell permeable GlcN-containing nucleotide and/or GlcN substrate willbecome cell membrane permeable upon conjugation to such a group L. Suchcell membrane transport enhancer groups L comprise, for example, alinear poly(arginine) of D- and/or L-arginine with 6-15 arginineresidues, linear polymers of 6-15 subunits which each carry aguanidinium group, oligomers or short-length polymers of from 6 to up to50 subunits, a portion of which have attached guanidinium groups, and/orparts of the sequence of the HIV-tat protein, in particular the subunitTat49-Tat57 (RKKRRQRRR in the one letter amino acid code).

Some labels L are spectroscopic probes and molecules which are one partof a specific binding pair that is capable of specifically binding to apartner (so-called affinity labels). Also, certain labels L aremolecules covalently attached to a solid support. Spectroscopic probesare optionally fluorophores. When the label L is a fluorophore, achromophore, a magnetic label, a radioactive label or the like,detection is by standard means adapted to the label and whether themethod is used in vitro or in vivo. Particular examples of labels L arealso radioactively labeled hexosamines.

Optionally, the labels are such that L of one GlcN-containing nucleotide(L1) is one member and L of a another GlcN-containing nucleotide or adifferently labeled nucleotide (L2) is the other member of twointeracting spectroscopic probes L1/L2, wherein energy can betransferred nonradiatively between the donor and acceptor (anotherfluorophore or a quencher) when they are in close proximity (less than10 nanometer distance) through either dynamic or static quenching. Anexample of such a pair of labels L1/L2 is a FRET (Förster (Fluorescence)resonance energy transfer).

Particular fluorophores considered are: Alexa Fluor dyes, includingAlexa Fluor® 350, 405, 430, 488, 514, 532, 546, 555, 568, 594, 610, 633,647, 660, 680, 700, 750, and 790 (Life Technologies, Carlsbad, Calif.);coumarin dyes, including 3-cyano-7-hydroxycoumarin,6,8-difluoro-7-hydroxy-4-methylcoumarin, 7-amino-4-methylcoumarin,7-ethoxy-4-trifluoromethylcoumarin, 7-hydroxy-4-methylcoumarin,7-hydroxycoumarin-3-carboxylic acid, 7-dimethylamino-coumarin-4-aceticacid, 7-amino-4-methyl-coumarin-3-acetic acid, and7-diethylamino-coumarin-3-carboxylic acid (Life Technologies, Carlsbad,Calif.); BODIPY® dyes, including BODIPY 493/503, FL, R6G, 530/550, TMR,558/568, 564/570, 576/589, 581/591, TR, 630/650, and 650/655 (LifeTechnologies, Carlsbad, Calif.); Quantum Dots, including Qdot® 545 ITK™,Qdot 565 ITK, Qdot 585 ITK, Qdot 605 ITK, Qdot 655 ITK, Qdot 705 ITK,and Qdot 800 ITK (Life Technologies, Carlsbad, Calif.); Oregon Green®dyes, including Oregon Green 488, 488, and 514 (Life Technologies,Carlsbad, Calif.); LanthaScreen® Tb Chelates (Life Technologies,Carlsbad, Calif.); Rhodamine 110, Rhodamine Green, Rhodamine Red, TexasRed-X, Cascade Blue, Pacific Blue, Marina Blue, Pacific Orange, DapoxylSulfonyl Chloride, Dapoxyl Carboxylic Acid, 1-Pyrenebutanoic Acid,1-Pyrenesulfonyl Chloride, 2-(2,3-Naphthalimino) ethylTrifluoromethanesulfonate, 2-Dimethylaminonaphthalene-5-SulfonylChloride, 2-Dimethylaminonaphthalene-6-Sulfonyl Chloride,3-Amino-3-Deoxydigoxigenin Hemisuccinamide,4-Sulfo-2,3,5,6-Tetrafluorophenol, 5-(and-6)-Carboxyfluorescein,5-(and-6)-Carboxynaphthofluorescein, 5-(and-6)-Carboxyrhodamine 6G,5-(and-6)-Carboxytetramethylrhodamine, 5-(and-6)-Carboxy-X-Rhodamine,5-Dimethylaminonaphthalene-1-Sulfonyl Chloride (Dansyl Chloride),6-((5-Dimethylaminonaphthalene-1-Sulfonyl)amino) Hexanoic Acid,Succinimidyl Ester (Dansyl-X, SE), Lissamine Rhodamine B SulfonylChloride, Malachite Green isothiocyanate, NBD Chloride,4-Chloro-7-Nitrobenz-2-Oxa-1,3-Diazole (4-Chloro-7-Nitrobenzofurazan),NBD Fluoride; 4-Fluoro-7-Nitrobenzofurazan,4-Fluoro-7-Nitrobenz-2-Oxa-1,3-Diazole, NBD-X, and PyMPO (LifeTechnologies, Carlsbad, Calif.); CyDyes®, including Cy 3, Cy 3B, Cy 3.5,Cy 5, Cy 5.5, Cy 7 (GE Healthcare, Little Chalfont, UK); ATTO dyes,including ATTO 390, 425, 465, 488, 495, 520, 532, 550, 565, 590, 594,610, 611X, 620, 633, 635, 637, 647, 647N, 655, 680, 700, 729, and 740(ATTO-TEC GmbH, Siegen, Germany); DY dyes, including DY 350, 405, 415,490, 495, 505, 530, 547, 548, 549, 550, 554, 555, 556, 560, 590, 591,594, 605, 610, 615, 630, 631, 632, 633, 634, 635, 636, 647, 648, 649,650, 651, 652, 654, 675, 676, 677, 678, 679, 680, 681, 682, 700, 701,703, 704, 730, 731, 732, 734, 749, 750, 751, 752, 754, 776, 777, 778,780, 781, 782, 800, 831, 480XL, 481XL, 485XL, 510XL, 520XL, and 521XL(Dyomics, Jena, Germany); CF™ dyes, including CF 350, 405, 485, 488,532, 543, 555, 568, 594, 620R, 633, 640R, 647, 660C, 660R, 680, 680R,750, 770, and 790 (Biotium, Hayward, Calif.); CAL Fluor® dyes, includingCAL Fluor Gold 540, Orange 560, Red 590, Red 610, Red 635 (BiosearchTechnologies, Novato, Calif.); Quasar® dyes, including Quasar 570, 670,705 (Biosearch Technologies, Novato, Calif.); Biosearch Blue and Pulsar®650 (Biosearch Technologies, Novato, Calif.); DyLight® Fluor dyes,including DYLight 350, 405, 488, 550, 594, 633, 650, 680, 755, 800(Thermo Fisher Scientific, Waltham, Mass.); FluoProbes® dyes, includingFluoProbes 390, 488, 532, 547H, 594, 647H, 682, 752, 782 (Interchim,Montlu çon Cedex, France); SeTau dyes, including SeTau 380, 425, 405,404, 655, 665, and 647 (SETA BioMedicals, Urbana, Ill.); Square dyes,including Square 635, 660, and 685 (SETA BioMedicals, Urbana, Ill.);Seta dyes, including, Seta 470, 555, 632, 633, 646, 650, 660, 670, 680,and 750 (SETA BioMedicals, Urbana, Ill.); SQ-565 and SQ-780 (SETABioMedicals, Urbana, Ill.); Chromeo™ dyes, including Chromeo 488, 494,505, 546, and 642 (Active Motif, Carlsbad, Calif.); Abberior STAR dyes,including STAR 440SX, 470SX, 488, 512, 580, 635, and 635P (AbberiorGmbH, Göttingen, Germany); Abberior CAGE dyes, including CAGE 500, 532,552, and 590 (Abberior GmbH, Göttingen, Germany); Abberior FLIP 565(Abberior GmbH, Göttingen, Germany); IRDye® Infrared Dye, includingIRDye 650, 680LT, 680RD, 700DX, 750, 800CW, and 800RS (LI-CORBiosciences, Lincoln, Nebr.); Tide Fluor™ dyes, including TF1, TF2, TF3,TF4, TF5, TF6, TF7, and TF8 (AAT Bioquest, Inc., Sunnyvale, Calif.);iFluor™ dyes, including iFluor™ 350, 405, 488, 514, 532, 555, 594, 610,633, 647, 680, 700, 750, and 790 (AAT Bioquest, Inc., Sunnyvale,Calif.); mFluor™ dyes, including mFluor Blue 570, Green 620, Red 700,Red780, Violet 450, Violet 510, Violet 540, and Yellow 630 (AATBioquest, Inc., Sunnyvale, Calif.); trFluor™ Eu and trFluor Tb (AATBioquest, Inc., Sunnyvale, Calif.); HiLyte Fluor™ dyes, including HiLyteFluor 405, 488, 555, 594, 647, 680, and 750 (AnaSpec, Inc., Fremont,Calif.); Terbium Cryptate and Europium Cryptate (Cisbio Bioassays,Codolet, France); and other nucleotide classical dyes, including FAM,TET, JOE, VIC™, HEX, NED, TAMRA, ROX™, Texas Red® (BiosearchTechnologies, Novato, Calif.).

Particular quenchers considered are: QSY 35, QSY 9, QSY 7, and QSY 21(Life Technologies Corporation, Carlsbad, Calif.); Black Hole Quencher®,including BHQ-0, BHQ-1, BHQ-2, and BHQ-3 (Biosearch Technologies, Inc.,Novato, Calif.); ATTO 540Q, ATTO 580Q, and ATTO 612Q (ATTO-TEC GmbH,Siegen, Germany); 4-dimethylamino-zobenzene-4′-sulfonyl derivatives(Dabsyl), 4-dimethylaminoazobenzene-4′-carbonyl derivatives (Dabcyl),DNP and DNP-X [6-(2,4-Dinitrophenyl)aminohexanoic acid] (AAT Bioquest,Inc., Sunnyvale, Calif. 94085, USA); DYQ quenchers, including DYQ 425,505, 1, 2, 660, 661, 3, 700, 4 (Dyomics, Jena, Germany); IRDye® QC-1(LI-COR Biosciences, Lincoln, Nebr.); Tide Quencher™, including TQ1,TQ2, TQ3, TQ4, TQ5, TQ6, and TQ7 (AAT Bioquest, Inc., Sunnyvale,Calif.); QXL® quenchers, including QXL 490, 570, 610, 670, and 680(AnaSpec, Inc., Fremont, Calif.); BlackBerry® Quenchers, includingBBQ-650 (Berry & Associates, Inc., Dexter, Mich.).

Other labels L are nonfluorescent but form fluorescent conjugatesstoichiometrically with amines. These reagents are particularly usefulfor detecting and quantitating amine-containing nucleotides. Examples ofsuch labels are fluorescamine, aromatic dialdehydes (e.g.,o-phthaldialdehyde (OPA) and naphthalene-2,3-dicarboxaldehyde (NDA)),ATTO-TAG CBQCA, ATTO-TAG FQ, 7-nitrobenz-2-Oxa-1,3-Diazole (NBD)derivatives, dansyl chloride, 1-pyrenesulfonyl chloride, dapoxylsulfonyl chloride, coumarins, pyrenes, and N-methylisatoic anhydride(Life Technologies Corporation, Carlsbad, Calif.).

Depending on the properties of the label L, the GlcN-containingnucleotide may be bound to a solid support on reaction with the GlcNmoiety. The label L may already be attached to a solid support whenentering into reaction with GlcN, or may subsequently, i.e. after GlcNis transferred to the nucleotide, be used to attach the labeledGlcN-containing nucleotide to a solid support. The label L may be onemember of a specific binding pair, the other member of which is attachedor attachable to the solid support, either covalently or by any othermeans. A specific binding pair considered is e.g. biotin and avidin orstreptavidin. Either member of the binding pair may be the label L ofthe substrate, the other being attached to the solid support. Examplesof specific binding pairs allowing covalent binding to a solid supportare e.g. SNAP-tag/AGT and benzylguanine derivatives (U.S. Pat. Nos.7,939,284; 8,367,361; 7,799,524; 7,888,090; and 8,163,479) or pyrimidinederivatives (U.S. Pat. No. 8,178,314), CLIP-tag/ACT and benzylcytosinederivatives (U.S. Pat. No. 8,227,602), Halotag and chloroalkenederivatives (Los, et al. Methods Mol Biol., 356:195-208 (2007)),serine-beta-lactamases and beta-lactam derivatives (International PatentApplication Publication No. WO 2004/072232). Further examples ofspecific binding pairs allowing covalent binding to a solid support areacyl carrier proteins and modifications thereof (binder proteins), whichare coupled to a phosphopantheteine subunit from Coenzyme A (bindersubstrate) by a synthase protein (U.S. Pat. No. 7,666,612). Examples oflabels allowing convenient binding to a solid support are e.g. chitinbinding domain (CBD), maltose binding protein (MBP), glycoproteins,transglutaminases, dihydrofolate reductases, glutathione-S-transferaseal (GST), FLAG tags, His-tags, or reactive substituents allowingchemoselective reaction between such substituent with a complementaryfunctional group on the surface of the solid support. Examples of suchpairs of reactive substituents and complementary functional group aree.g. amine and activated carboxy group forming an amide, azide and apropiolic acid derivative undergoing a 1,3-dipolar cycloadditionreaction, amine and another amine functional group reacting with anadded bifunctional linker reagent of the type of activatedbis-dicarboxylic acid derivative giving rise to two amide bonds, orother combinations known in the art. Examples of a convenient solidsupport are e.g. glass surfaces such as glass slides, microtiter plates,and suitable sensor elements, in particular functionalized polymers(e.g. in the form of beads), chemically modified oxidic surfaces, e.g.silicon dioxide, tantalum pentoxide or titanium dioxide, or alsochemically modified metal surfaces, e.g. noble metal surfaces such asgold or silver surfaces.

When the label L is capable of generating reactive radicals, such ashydroxyl radicals, upon exposure to an external stimulus, the generatedradicals can then inactivate proteins that are in close proximity of theGlcN-containing nucleotide, allowing studying the role of theseproteins. Examples of such labels are tethered metal-chelate complexesthat produce hydroxyl radicals upon exposure to H₂O₂ and ascorbate, andchromophores such as malachite green that produce hydroxyl radicals uponlaser irradiation. The use of chromophores and lasers to generatehydroxyl radicals is also known in the art as chromophore assisted laserinduced inactivation (CALI). Furthermore, proteins which are in closeproximity of the GlcN-containing nucleotide can be identified as such byeither detecting fragments of that protein by a specific antibody, bythe disappearance of those proteins on a high-resolution2D-electrophoresis gels or by identification of the cleaved proteinfragments via separation and sequencing techniques such as massspectrometry or protein sequencing by N-terminal degradation.

When the label L is a molecule that can cross-link to other nucleicacids or proteins, e.g. a molecule containing functional groups such asmaleimides, active esters, or azides and others known to thoseproficient in the art, contacting such a labeled GlcN-containingnucleotide that interacts with other nucleic acids or proteins leads tothe covalent cross-linking of the GlcN-containing nucleotide with itsinteracting nucleic acid or protein via the label. This allows theidentification of the nucleic acid or protein interacting with theGlcN-containing nucleotide. In a special aspect of cross-linking, thelabel L is a molecule which enables photo-reactive (light-activated)chemical crosslinking. Labels L for photo cross-linking are e.g.benzophenones, phenyl azides, and diazirines.

Other labels L considered are for example fullerenes, boranes forneutron capture treatment, nucleotides or oligonucleotides, e.g. forself-addressing chips, peptide nucleic acids, and metal chelates, e.g.platinum chelates that bind specifically to DNA. A particularbiomolecule with desirable enzymatic, chemical or physical properties ismethotrexate. Methotrexate is a tight-binding inhibitor of the enzymedihydrofolate reductase (DHFR) and can be used with the well-known classof so-called “chemical inducers of dimerization” (CIDs).

All references cited herein are incorporated by reference.

EXAMPLES Example 1 Evaluation of UDP-GlcN and UDP-Glc Derivatives forSelective Labeling of 5-hmC Materials and Methods Enzymes, UDP-GlcN andUDP-Glc Derivatives, and DNA Preparation.

General procedures and reagents for the synthesis of UDP-GlcN andUDP-Glc derivatives are provided in Example 2. The BGT, BGT/Y261L, andAGT, and the restriction endonucleases MfeI, XhoI, and TaqI wereobtained from New England Biolabs, Inc. (Ipswich, Mass.). BGT DNA waspurified following the protocol adapted from Ehrlich et al. [Ehrlich,M., Ehrlich, K. and Mayo, J. A. (1975) Unusual properties of the DNAfrom Xanthomonas phage XP-12 in which 5-methylcytosine completelyreplaces cytosine. Biochim Biophys Acta, 395, 109-119]. Fluorescein(FAM) labeled double stranded oligonucleotides containing a single 5-hmCresidue were synthesized as described by Kinney et al. [Kinney, S. M.,Chin, H. G., Vaisvila, R., Bitinaite, J., Zheng, Y., Esteve, P. O.,Feng, S., Stroud, H., Jacobsen, S. E. and Pradhan, S. (2011)Tissue-specific distribution and dynamic changes of 5-hmC in mammaliangenomes. J Bio Chem, 286, 24685-24693]. The final 49-bp dsoligonucleotide sequence is as follows: top strand-5′-/FAM/-CTACACCCATCACATTTACCT^(5hm)CGAGTAAGAGTTGATAGTAGAGTTGAGA-3′(SEQ ID NO:1); and bottom strand-3′-GATGTGGGTAGTGTAAATGGAGCTCATTCTCAACTATCATCTCAACTCT-/FAM/-5′ (SEQ IDNO:2); (XhoI/TaqI site is underlined).

Glycosylation of BGT DNA with Wild-Type AGT, Wild-Type BGT, andBGT/Y261L in the Presence of UDP-GlcN and UDP-Glc Derivatives.

5-hmC residues were glycosylated by incubating 0.25 μg BGT DNA substratewith 100 ng of wild-type AGT, wild-type BGT, or BGT/Y261L for 1 h at 37°C. in 15 μL reaction containing 1X NEBuffer 4 supplemented with variousconcentrations of UDP-Glc derivatives (ranging from 1.0 μM to 5.0 mM).After glycosylation, the glucosyltransferase was heat-inactivated byincubating for 10 minutes at 70° C. To analyze the enzyme-catalyzedtransfer efficiency, samples were incubated with 1 μL (20 units) MfeIfor 1 hour at 37° C. Reaction products were separated by agarose gelelectrophoresis and stained with ethidium bromide to check for completeprotection of BGT DNA against MfeI cleavage.

Inhibitory Tests of Wild-Type BGT and BGT/Y261L with UDP-2-Azido-Glc.

0.25 μg BGT DNA was combined with various concentrations ofUDP-2-Azido-Glc 2 (from 0.02 to 10.0 mM) in the presence of 40 μM nativeUDP-Glc cofactor (New England Biolabs, Ipswich, Mass.), or combined withvarious concentrations of natural UDP-Glc (from 0.02 to 20.0 mM) in thepresence of 5 mM UDP-2-Azido-Glc 2. After adding 100 ng of wild-type BGTor BGT/Y261L, the reaction mixtures were incubated at 37° C. for 1 hourin 1X NEBuffer 4. After glycosylation, the glucosyltransferase washeat-inactivated by incubating for 10 minutes at 70° C. To analyze theinhibitory effect of UDP-2-Azido-Glc 2, samples were incubated with 1 μL(20 units) MfeI for 1 hour at 37° C. Reaction products were separated byelectrophoresis on agarose gel and stained with ethidium bromide.

Glycosylation of 5-hmC-Containing Synthetic Oligonucleotides.

5-hmC residues were glycosylated by incubating 1 nmol of a 49-bpFAM-labeled double-stranded oligonucleotide substrate with 1 μg of BGTfor 18 hours at 37° C. in a total 100 μL reaction containing 50 mM HEPESbuffer pH 7.6, 10 mM MgCl₂, 50 mM NaCl and supplemented with 0.5 mMmodified UDP-glucose derivative. After glycosylation, theoligonucleotide was purified by phenol-chloroform extraction and ethanolprecipitation and dissolved in 50 μL H₂O. To ensure complete 5-hmCglycosylation, a 10 pmol aliquot was digested with either 1 μL (20units) of XhoI or TaqI restriction endonuclease for 1 hour at 37° C. Forinstance, XhoI cuts 5-hmC residues, but does not cut 5-gmC residuesresulting from incubation with UDP-6-GlcN; TaqI cuts both 5-hmC and5-gmC residues (FIG. 9). Reaction products were separated byelectrophoresis under non-denaturing conditions on a 10-20% acrylamidegel and visualized using a Typhoon™ 9400 imager (GE Healthcare, LifeSciences, Piscataway, N.J.) with a standard fluorescein filter set(488/526 nm excitation/emission).

Click Chemistry Labeling.

50 pmol of a 6-Azido-Glc-containing 49-bp FAM-labeled oligonucleotidewas incubated with the indicated concentrations ofdibenzylcyclooctyne-tetramethylrhodamine (DBCO-Fluor 545, ClickChemistry Tools, Scottsdale, Ariz.) in a total 10 μL reaction for 18hours at room temperature or for 8 hours at 37° C. Unincorporatedfluorophore was removed by gel-filtration on a microspin G-50 column. Toanalyze the labeling efficiency, 10 pmol samples were separated byelectrophoresis under non-denaturing conditions on a 10%-20% acrylamidegel and visualized using a Typhoon 9400 imager with standard fluorescein(488/526 nm excitation/emission) and rhodamine (532/580 nmexcitation/emission) filter sets.

Activated Ester Labeling.

50 pmol of 2- or 6-GlcN-containing 49-bp FAM-labeled oligonucleotideswere incubated with the indicated concentrations of5-(and-6)-carboxytetramethylrhodamine succinimidyl ester (TAMRA NHSester, Life Technologies, Carlsbad, Calif.) in 50 mM HEPES buffer pH 7.6in a total 10 μL reaction at room temperature for 18 hours.Unincorporated fluorophore was removed by gel-filtration on a microspinG-50 column. Electrophoresis was performed on a 10-20% acrylamide gel tocheck the labeling efficiency. To optimize the coupling conditions, 50pmol of 6-GlcN-containing 49-bp FAM-labeled oligonucleotide wasincubated with 5.0 mM TAMRA NHS ester in different buffers ranging frompH 5.5 to 8.4 as indicated. Unincorporated fluorophore was removed bygel-filtration on a microspin G-50 column. To analyze the labelingefficiency, a 10 pmol aliquot was digested with 1 μL (20 units) TaqIrestriction endonuclease for 1 hour at 37° C. Reaction products wereseparated by electrophoresis under non-denaturing conditions on a10%-20% acrylamide gel and visualized using a Typhoon 9400 imager withstandard fluorescein (488/526 nm excitation/emission) and rhodamine(532/580 nm excitation/emission) filter sets.

Results Synthesis of UDP-GlcN and UDP-Glc Derivatives.

Referring now to FIG. 2, BGT can be used to add a glucosamine or amodified glucose derivative to the hydroxyl group of 5-hmC using aUDP-Glc derivative as a substrate (FIG. 2A). The available X-ray crystalstructure of UDP-Glc/wild-type BGT complex [Lariviere, L.,Gueguen-Chaignon, V. and Morera, S. (2003) Crystal structures of the T4phage beta-glucosyltransferase and the D100A mutant in complex withUDP-glucose: glucose binding and identification of the catalytic basefor a direct displacement mechanism. J. Mol. Biol., 330, 1077-1086]shows that the more conservative O3′ and O4′ hydroxyl groups of UDP-Glcparticipate in multiple hydrogen bonds within the binding pocket of theenzyme, while O2′ and O6′ hydroxyl groups are only involved in onehydrogen bond each. Accordingly, it was postulated O2′ and O6′ hydroxylwould be the most suitable positions for the installation of chemicaltags (e.g., including, but not limited to, azido, amino, or ketone) fordetection and enrichment of 5-hmC residues in double-stranded DNA (FIG.2A). Azido, amino, and ketone functional groups have been widely usedfor in vitro conjugation of large biomolecules under physiologicalconditions [Sletten, E. M. and Bertozzi, C. R. (2009) Bioorthogonalchemistry: fishing for selectivity in a sea of functionality. Angew ChemInt Ed Engl, 48, 6974-6998]. Azido groups can orthogonally react withalkyne functionalized probes via Huisgen-Sharpless azide-alkyne [3+2](also known as click chemistry) or strain-promoted cycloaddition. Aminogroups can specifically react with active esters, such astetrafluorophenyl (TFP) and N-hydroxysuccinimidyl (NHS) esters. Ketonegroups can be selectively modified with hydrazine or hydroxylaminonucleophiles.

The UDP-glucose derivative UDP-6-Azido-Glc 1 was previously demonstratedto be a substrate for the wild-type BGT, showing about a 6-fold decreasein the reaction rate compared to the native UDP-Glc cofactor [Song, C.X., Szulwach, K. E., Fu, Y., Dai, Q., Yi, C., Li, X., Li, Y., Chen, C.H., Zhang, W., Jian, X. et al. (2011) Selective chemical labelingreveals the genome-wide distribution of 5-hydroxymethylcytosine. Nat.Biotechnol., 29, 68-72]. Thus, UDP-6-Azido-Glc 1 was used as acomparative model in these studies. UDP-2-Azido-Glc 2 was synthesizedusing a similar protocol [Song, C. X., Szulwach, K. E., Fu, Y., Dai, Q.,Yi, C., Li, X., Li, Y., Chen, C. H., Zhang, W., Jian, X. et al. (2011)Selective chemical labeling reveals the genome-wide distribution of5-hydroxymethylcytosine. Nat. Biotechnol., 29, 68-72; Marchesan, S, andMacmillan, D. (2008) Chemoenzymatic synthesis of GDP-azidodeoxymannoses:non-radioactive probes for mannosyltransferase activity. Chem. Commun.,4321-4323].

UDP-glucosamine cofactors UDP-6-GlcN 3 and UDP-2-GlcN 4 were synthesizedby hydrogenation with H₂ on Pd/C from 1 and 2, respectively (FIG. 3)[Takaya, K., Nagahori, N., Kurogochi, M., Furuike, T., Miura, N., Monde,K., Lee, Y. C. and Nishimura, S. (2005) Rational design, synthesis, andcharacterization of novel inhibitors for humanbeta-1,4-galactosyltransferase. J. Med. Chem., 48, 6054-6065].UDP-2-Keto-Glc 5 was synthesized according to a previously reportedmethod [Dulcey, A. E., Qasba, P. K., Lamb, J. and Griffiths, G. L.(2011) Improved synthesis of UDP-2-(2-ketopropyl)galactose and a firstsynthesis of UDP-2-(2-ketopropyl)glucose for the site-specific linkingof biomolecules via modified glycan residues using glycosyltransferases.Tetrahedron, 67, 2013-2017; Boeggeman, E., Ramakrishnan, B. and Qasba,P. K. (2010), Annual Conference of the Society for Glycobiology, St PeteBeach, Fla., Vol. 20, pp. 1511]. The final products were purified with aDevelosil RP Aqueous C30 semi-preparative column by HPLC, and theiridentities and purities were confirmed by LC-MS and high-resolution massspectrometry.

Enzymatic Transfer of UDP-GlcN and UDP-Glc Derivatives to BGT DNA.

T4 phage α- and β-glucosyltransferases (AGT and BGT, respectively)specifically transfer the glucose moiety of UDP-Glc to 5-hmC residues inds DNA, making α- and β-glucosyloxy-5-methylcytosine (5-gmC)[Tomaschewski, J., Gram, H., Crabb, J. W. and Ruger, W. (1985)T4-induced alpha- and beta-glucosyltransferase: cloning of the genes anda comparison of their products based on sequencing data. Nucleic AcidsRes., 13, 7551-7568; Szwagierczak, A., Bultmann, S., Schmidt, C. S.,Spada, F. and Leonhardt, H. (2010) Sensitive enzymatic quantification of5-hydroxymethylcytosine in genomic DNA. Nucleic Acids Res., 38, e181].The transfer efficiency of compounds 1-5 by AGT, BGT, and BGT/Y261Lmutant [Boeggeman, E., Ramakrishnan, B. and Qasba, P. K. (2010), AnnualConference of the Society for Glycobiology, St Pete Beach, Fla., Vol.20, pp. 1511] was assessed by incubating various concentrations of eachcompound with BGT DNA (FIGS. 4 and 5).

BGT DNA contains 5-hmC instead of cytosine and was produced from phagedeficient in α-and β-glucosyltransferases. The degree of glycosylationof BGT DNA was determined by incubating the samples with the restrictionendonuclease MfeI. Because MfeI cleaves ^(5-hm)C↓AATTG sites but isblocked by their glycosylation, this restriction enzyme was used toquantify the transfer efficiency of each of the UDP-GlcN and UDP-Glcderivatives by the glucosyltransferases.

FIG. 4 shows the results of enzymatic transfer experiments with (A)natural UDP-Glc, (B) UDP-6-Azido-Glc 1, (C) UDP-6-GlcN 3, and (D)UDP-2-GlcN 4. Top row: wild-type BGT; middle row: BGT/Y261L; bottom row:wild-type AGT. The vertical parallel lines show the minimumconcentration for complete protection of BGT DNA against MfeI cleavage,which cuts hydroxymethylated cytosine (hmC) at ^(hm)CAATTG. Variousconcentrations of UDP-Glc derivatives (as indicated) were incubated with0.25 μg BGT DNA in the presence of 100 ng of glucosyltransferase at 37°C. for 1 hour. Then the glucosyltransferase was killed by heat at 70°C., and 1 μL (20 units) MfeI was added and incubated at 37° C. for 1hour. The results were analyzed by agarose gel electrophoresis.Concentration values are given in mM.

FIG. 5 shows the results of enzymatic transfer experiments with (A)UDP-2-Azido-Glc 2, (B) UDP-2-Keto-Glc 5, (C) UDP-GlcUA, and (D)UDP-GlcNAc. Top row: wild-type BGT; middle row: BGT/Y261L; bottom row:wild-type AGT. The vertical parallel lines show the minimumconcentration for complete protection of BGT DNA against MfeI cleavage,which cuts hmC at ^(hm)CAATTG. Various concentrations of each substrate(as indicated) were incubated with 0.25 μg BGT DNA in the presence of100 ng glucosyltransferase at 37° C. for 1 hour. Then theglucosyltransferase was killed by heat at 70° C. for 10 minutes, and 1μL (20 units) MfeI was added and incubated at 37° C. for 1 hour. Theresults were analyzed by agarose gel electrophoresis. Concentrationvalues are given in mM.

FIG. 6 shows the results of UDP-2-Azido-Glc 2 inhibitory tests withwild-type BGT (top row) and BGT/Y261L (bottom row). FIG. 6A: Variousconcentrations of UDP-2-Azido-Glc 2 (as indicated) in the presence of 40μM underivatized UDP-Glc. The vertical parallel lines indicate theminimum concentration of UDP-2-Azido-Glc 2 in which BGT DNA is notprotected against MfeI cleavage, demonstrating that the glucose transferis inhibited. FIG. 6B: Various concentrations of natural UDP-Glc (asindicated) in the presence of 5 mM UDP-2-Azido-Glc 2. The verticalparallel lines indicate the minimum concentration of UDP-2-Azido-Glc 2for complete protection of BGT DNA against MfeI cleavage. Concentrationvalues are given in mM. The results show that UDP-2-Azido-Glc 2 is acompetitive inhibitor for wild-type BGT and BGT/Y261L.

UDP-6-Azido-Glc 1, UDP-6-GlcN 3, and UDP-2-GlcN 4 were transferred bywild-type BGT with similar efficiency as the natural UDP-Glc (FIG. 4).None of the synthesized UDP-GlcN or UDP-Glc derivatives could beefficiently transferred by wild-type AGT. AGT and BGT proteins shareabout 20% sequence similarity, but differ in the formation of theglycosidic linkage: AGT is a retaining, whereas BGT is an invertingglycosyltransferase.

BGT can glycosylate all available 5-hmC bases in acceptor DNA; however,AGT has some restrictions and its precise substrate specificity is notyet fully understood [Tomaschewski, J., Gram, H., Crabb, J. W. andRuger, W. (1985) T4-induced alpha- and beta-glucosyltransferase: cloningof the genes and a comparison of their products based on sequencingdata. Nucleic Acids Res., 13, 7551-7568]. Interestingly, the mutantBGT/Y261L transferred UDP-2-GlcN 4 with similar efficiency as naturalUDP-Glc, but the transfer efficiency for UDP-6-GlcN 3 was about 15-foldlower. According to Qasba et al., the side chain of Tyr261 hinders thebinding of the N-acetyl-group of UDP-GlcNAc allowing only UDP-Glc to bethe donor substrate; consequently, the Y261L mutation enhances theenzyme activity towards GlcNAc or glucose with a chemical tag at the C2position [Boeggeman, E., Ramakrishnan, B. and Qasba, P. K. (2010),Annual Conference of the Society for Glycobiology, St Pete Beach, Fla.,Vol. 20, pp. 1511]. In fact, it was observed that BGT/Y261L transfersUDP-GlcNAc 10-fold more efficiently than the wild-type BGT (FIG. 5).However, compared to UDP-2-GlcN 4, which lacks the N-acetyl-group, thetransfer efficiency of UDP-GlcNAc by the Y261L enzyme is approximately100-fold lower. Furthermore, the transfer of UDP-2-Keto-Glc 5 byBGT/Y261L was not detected at the highest tested concentration (4 mM).

None of the three tested enzymes efficiently transferred UDP-2-Azido-Glc2 to 5-hmC residues in BGT DNA (FIG. 5). UDP-2-Azido-Glc acts as acompetitive inhibitor for wild-type BGT and BGT/Y261L (FIG. 6A), but notfor AGT. Likewise, with the increase of natural UDP-Glc concentration,UDP-2-Azido-Glc could be chased out to restore the activity of theβ-glucosyltransferase (FIG. 6B). The transfer efficiencies of thesynthesized UDP-Glc derivatives 1-5, along with the native UDP-Glccofactor and the commercially available UDP-sugars, UDP-Glucuronic acid(UDP-GlcUA) and UDP-N-acetyl-D-glucosamine (UDP-GlcNAc), are presentedin Table 1.

TABLE 1 UDP-Sugar concentration required for complete protection of BGTDNA from MfeI cleavage. Concentration of UDP-Sugar (mM) UDP- UDP- UDP-Enzyme Glc GlcUA GlcNAc 1 2 3 4 5 BGT 0.02 1.0 25 0.04 n.a. 0.020.03 >>4.0 BGT/Y261L 0.02 >>10.0 2.5 >>1.0 n.a. 0.31 0.03 >>4.0 AGT0.02 >>20.0 >>20.0 n.a. n.a. >>5.0 >>1.25 n.a. >>incomplete protectionevent at the highest tested concentration; n.a.: no activity; UDP-Glc:UDP-Glucose; UDP-GlcUA: UDP-Glucuronic acid; UDP-GlcNAc:UDP-N-acetyl-D-glucosamine; 1: UDP-6-Azido-Glc; 2: UDP-2-Azido-Glc; 3:UDP-6-glucosamine, UDP-6-GlcN; 4: UDP-2-glucosamine, UDP-2-GlcN; 5:UDP-2-Keto-Glc.

Without wishing to be bound by any theory, the size and polarity of thegroup at the O2′ position may be comparatively more important for thecatalytic activity of BGT. Site-specific mutations around the O6′binding area may enable mutant BGT enzymes to transfer larger molecules,such as UDP-GlcN or UDP-Glc derivatives with covalently bound reportergroups such as fluorophores or biotin.

It is surprising that BGT/Y261L cannot efficiently transferUDP-6-Azido-Glc. Without wishing to be bound by any theory, it isbelieved that the concurrent polarity change at the O2′ and O6′positions may result in the structural alterations of the enzyme bindingpocket, which may cause inactivation of the enzyme.

Fluorescent Labeling of Glucosamine-Modified Oligonucleotides.

Next, the labeling of 2- and 6-glucosamine-modified 5-hmC residues withcommercially available fluorescent probes was investigated, and theresults with the 6-azido-glucose DNA template were compared. To thisend, a synthetic 49-mer 5′-FAM-labeled duplex oligonucleotide containinga single 5-hmC residue [Kinney, S. M., Chin, N. G., Vaisvila, R.,Bitinaite, J., Zheng, Y., Esteve, P. O., Feng, S., Stroud, H., Jacobsen,S. E. and Pradhan, S. (2011) Tissue-specific distribution and dynamicchanges of 5-hydroxymethylcytosine in mammalian genomes. J Bio Chem,286, 24685-24693] was first incubated with wild-type BGT and thecompounds UDP-6-Azido-Glc 1, UDP-6-GlcN 3, or UDP-2-GlcN 4, to generatethe corresponding 6-azido-glucosyloxymethylcytosine,6-glucosaminyloxymethylcytosine and 2-glucosaminyloxymethylcytosineoligonucleotides. After purification by phenol extraction followed byethanol precipitation, the azido-glucose- and glucosamine-containingoligonucleotides were chemically labeled with an orthogonal fluorescentprobe via click chemistry (for 1) or activated ester coupling (for 3 and4) (FIG. 7).

FIG. 7 shows a schematic for the two-step detection of 5-hmC residues ina 5′-FAM-labeled duplex oligonucleotide with reporter probes: (1) BGTmediated transglycosylation reaction using UDP-glucosamine orUDP-glucose and (2) Chemical labeling of resulting glucosamine- orazido-sugar-containing duplex oligonucleotide with fluorescent probes.TaqI restriction endonuclease cleaves the sequence T↓CGA at the positionindicated by the arrow.

For the click chemistry coupling, 6-Azido-Glc-modified oligonucleotide(5 μM) was incubated with 0.01 to 1.0 mM ofdibenzylcyclooctyne-tetramethylrhodamine (DBCO-Fluor 545) at roomtemperature for 18 hours. For the activated ester coupling, 2- or6-glucosamine-modified oligonucleotides (5 μM) were incubated withvarious concentrations of TAMRA NHS ester, ranging from 1.0 mM to 5.0mM, in 20 mM phosphate buffer pH 6.8 at room temperature for 18 hours.The coupling efficiency of each coupling reaction was analyzed using ascanning fluorometer after electrophoretic separation of products on a10-20% acrylamide gel (FIG. 8). To allow a better distinction betweenlabeled and unlabeled bands, samples were digested with TaqI restrictionendonuclease (FIG. 9). Because the cyclooctyne-based linker led to asubstantial band shift, the click chemistry coupling reactions could bedirectly visualized on gel without the need for restriction digestion.

FIG. 8 shows the results of labeling tests of (A) 6-Azido-Glc- and (B)6-GlcN-containing synthetic oligonucleotides. For copper-free clickcoupling, 50 pmols FAM-labeled 49-bp synthetic oligonucleotidecontaining a single 6-Azido-Glc were incubated with the indicatedconcentrations of DBCO-Fluor 545 (in mM) at room temperature for 18hours. For NHS-ester coupling, 50 pmols FAM-labeled 49-bp syntheticoligonucleotide containing a single 6-glucosamine were incubated withthe indicated concentrations of TAMRA NHS ester (in mM) in 20 mMphosphate buffer pH 6.8 at room temperature for 18 hours, andsubsequently cleaved with 1 μL (20 units) TaqI restriction endonucleasefor 1 hour at 37° C. in order to separate labeled from unlabeledoligonucleotides. Top row: FAM channel (488 nm); middle row: TAMRAchannel (532 nm); bottom row: overlay.

FIG. 9 shows the results of restriction endonuclease cleavage of 5-hmC-and 5-gmC-containing oligonucleotides. FAM-labeled 49-bp syntheticoligonucleotide (1 nmol) was glycosylated with 100 ng of BGT for 18hours at 37° C. in a total 100 mL reaction containing 50 mM HEPES bufferpH 7.6, 10 mM MgCl₂, 50 mM NaCl supplemented with 0.5 mM UDP-6-GlcN.Aliquots (10 pmol) were digested for 1 hour at 37° C. either with 1 μL(20 units) of XhoI or TaqI before (lanes 1-3) and after (lanes 4-6)glycosylation. XhoI restriction endonuclease cleaves CT5-^(hm)CGACsites, but is blocked by 5-^(hm)C glycosylation. TaqI cleaves bothT5-^(hm)CGA and T5-^(gm)CGA sites.

FIG. 10 shows the results of labeling of a 6-Azido-Glc-modifiedoligonucleotide at different temperatures. FAM-labeled 49-bp syntheticoligonucleotide (50 pmols) containing a single 6-Azido-Glc was incubated1 mM DBCO-Fluor 545 for 0, 2, 4, 8 and 18 h at (A) room temperature or(B) at 37° C. Top row: FAM channel (488 nm); middle row: TAMRA channel(532 nm); bottom row: overlay.

FIG. 11 shows the results of labeling of 6-glucosamine- and2-glucosamine-modified oligonucleotides. FAM-labeled 49-bp syntheticoligonucleotide (50 pmols) containing either a single 6-glucosamine or a2-glucosamine were incubated with different concentrations of TAMRA NHSester (as indicated) in HEPES buffer pH 7.6 at room temperature for 18hours. Top row: FAM channel (488 nm); middle row: TAMRA channel (532nm); bottom row: overlay. Concentration values are given in mM. (A)Uncut oligonucleotides; (B) Oligonucleotides cleaved by TaqI. TaqItreatment generates two smaller ds oligonucleotides and allows for aclearer distinction among labeled and unlabeled 5-gmC residues. Labelingefficiencies were estimated at the FAM channel by comparing fluorescenceintensity from cut oligonucleotide bands.

FIG. 12 shows the results of labeling of 6-glucosamine-modifiedoligonucleotides at different pHs. FAM-labeled 49-bp syntheticoligonucleotide (50 pmols) containing a single 6-glucosamine wereincubated with 5.0 mM TAMRA NHS ester in 20 mM phosphate buffer with pHsranging from 5.6-7.6 at room temperature for 18 hours. The resultingoligonucleotides were cleaved with TaqI before gel electrophoresis. Toprow: FAM channel (488 nm); middle row: TAMRA channel (532 nm); bottomrow: overlay. The results show that the best pH range for NHS esterlabeling is between pH 6.8-7.4.

FIG. 13 shows the results of labeling of 6-glucosamine-modifiedoligonucleotides at different temperatures. Various concentrations ofFAM-labeled 49-bp synthetic oligonucleotide containing a single6-glucosamine (as indicated) were incubated with 5.0 mM TAMRA NHS esterin 20 mM pH 6.8 phosphate buffer for 18 hours at (A) room temperature or(B) 37° C. The resulting oligonucleotides were cleaved with TaqI beforegel electrophoresis. Top row: FAM channel (488 nm); middle row: TAMRAchannel (532 nm); bottom row: overlay. Concentration values are given inmM. The results show that increasing temperature doesn't significantlyaffect the NHS ester labeling efficiency.

The results show that for the click coupling, 1 mM DBCO-Fluor 545 wassufficient to label 5 μM single 6-Azido-Glc-modified oligonucleotidewithin 18 hours at room temperature (FIG. 8A) or 8 hours at 37° C. (FIG.10) with close to 100% labeling yield. For the activated ester method, 5mM TAMRA NHS ester was required to efficiently label 5 μM single6-glucosamine-modified oligonucleotide within 18 h at room temperatureat the optimized pH of 6.8 (FIGS. 8B, 11, 12, and 13). The labelingratio for TAMRA NHS coupling with the 6-glucosamine-modifiedoligonucleotide ranged from 80%-90%.

Labeling DNA with glucosamine offers some unique advantages. Forexample, by combination of commercially available anti-glucosamineantibodies and amine-reactive fluorogenic tags, such asnaphthalene-2,3-dicarboxaldehyde (NDA) and fluorescamine [Wang, W.,Maniar, M., Jain, R., Jacobs, J., Trias, J. and Yuan, Z. (2001) Afluorescence-based homogeneous assay for measuring activity ofUDP-3-O—(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase. AnalBiochem, 290, 338-346; Skelley, A. M. and Mathies, R. A. (2006) Rapidon-column analysis of glucosamine and its mutarotation by microchipcapillary electrophoresis. J Chromatogr A, 1132, 304-309; Novatchev, N.and Holzgrabe, U. (2002) Evaluation of amino sugar, low molecularpeptide and amino acid impurities of biotechnologically produced aminoacids by means of CE. J Pharm Biomed Anal, 28, 475-486], 5-hmCenrichment and identification methods can be developed and may findpractical application in epigenetic research. Furthermore, the chemicalorthogonality of the azido and amino labeling systems opens up thepossibility to simultaneously label 5-hmC residues with differentsynthetic probes using engineered glycosyltransferases capable ofselectively transferring either a glucosamine or an azido-modifiedglucose moiety.

In summary, a new method for labeling 5-hmC residues in duplex DNA withfluorescent or affinity probes has been described. In some embodiments,the method involves using a UDP-GlcN cofactor, where small amino groupsare amenable for indirect labeling, and transferring these glucosaminemoieties using the transglycosylation reaction mediated by T4 phageglucosyltransferases. The labeling of glucosamine-containing DNA canoptionally be achieved using activated NHS-esters to install the desiredreporter group on DNA for downstream applications, such aslocus-specific detection 5-hmC or selective enrichment of 5-hmC bystandard affinity purification protocols. Equipping 5-hmC residues inDNA molecules with reporter probes can elucidate not only the 5-hmCdistribution in the genome, but also its temporal fluctuation andmaintenance, both in healthy and diseased states, as well as how it isinfluenced by local environment changes and the biology of eachindividual.

Example 2 Synthesis of UDP-GlcN and UDP-Glc Derivatives Materials

Reagents were purchased from Sigma-Aldrich; solvents were purchased fromFisher unless otherwise noted. Anhydrous solvents were purchased fromACROS, and used directly from sealed bottles, which were stored underargon. Brine (NaCl), NaHCO₃, and NH₄Cl refer to saturated aqueoussolutions unless otherwise noted. Silica column chromatography wasperformed with 32-63 μm silica gel and reagent grade solvents.Analytical LC-MS was performed on a Waters X-Bridge C18 reverse phasecolumn (2.5 μm, 4.6×75 mm) or a Develosil C30 RP Aqueous reverse phasecolumn (5 μm, 4.6×150 mm) with H₂O and CH₃CN as mobile phases on anAgilent 1200 HPLC system equipped with Agilent 6120 Quadrupole MassDetector at both positive and negative mode. Preparative HPLCpurification was performed on a Waters X-Bridge C18 reverse phase column(5 μm, 10×150 mm) or a Develosil RP Aquesous C30 column (5 μm, 10×150mm) with 100 mM pH 7.0 TEAB and CH3CN as mobile phases on an Agilent1200 HPLC system, unless otherwise noted. UV gel pictures were taken onan AlphaImager HP gel imaging system. Fluorescence gel pictures weretaken on a GE Health Care Typhoon 9400 high performance gel and blotimager at 488 nm (FAM, green false color) or 532 nm (TAMRA, red falsecolor).

Methods

Synthesis of UDP-6-Azido-Glc (FIG. 2B; compound 1) and UDP-2-Azido-Glc(FIG. 2B; compound 2)

UDP-6-Azido-Glc 1 and UDP-2-Azido-Glc 2 were synthesized from6-Azido-6-deoxy-D-glucose (Carbosynth #MA02620, Compton, Berkshire, UK)and 1,3,4,6-Tetra-O-acetyl-2-azido-2-deoxy-β-D-glucopyranose (TCIAmerica, # T2196, Portland, Oreg.), respectively, according to publishedmethods [Marchesan, S, and Macmillan, D. (2008) Chemoenzymatic synthesisof GDP-azidodeoxymannoses: non-radioactive probes formannosyltransferase activity. Chem. Commun., 4321-4323; Song, C. X.,Szulwach, K. E., Fu, Y., Dai, Q., Yi, C., L1, X., L1, Y., Chen, C. H.,Zhang, W., Jian, X. et al. (2011) Selective chemical labeling revealsthe genome-wide distribution of 5-hydroxymethylcytosine. Nat.Biotechnol., 29, 68-72] and their identities and purities were confirmedby HRMS, and analytical HPLC, respectively. UDP-6-Azido-Glc 1: HRMS(m/z): [M-H]-calculated for C₁₅H₂₂N₅O₁₆P₂, 590.0542; observed, 590.0545.UDP-2-Azido-Glc 2: HRMS (m/z): [M-H]-calculated for C₁₅H₂₂N₅O₁₆P₂,590.0542; observed, 590.0548.

Synthesis of UDP-6-GlcN (FIG. 2B; compound 3).

UDP-6-Azido-Glc 1 (37 mg, 0.062 mmol) was dissolved in 15 mL H₂O, and10% Pd—C (40 mg) was added. The reaction mixture was stirred under 1 atmH2 at room temperature for 45 minutes. Then the resulting solution wasfiltered through 0.22 μm membrane, concentrated by lyophilization, andthen purified with HPLC on a Develosil C30 RP Aqueous reverse phasecolumn. The final product was obtained as a white solid (30 mg, 85%).HRMS (m/z): [M-H]-calculated for C₁₅H₂₄N₃O₁₆P₂, 564.0637; observed,564.0641.

Synthesis of UDP-2-GlcN (FIG. 2B; Compound 4).

UDP-2-Azido-Glc 2 (17 mg, 0.027 mmol) was dissolved in 8 mL H2O, and 10%Pd—C (25 mg) was added. The reaction mixture was stirred under 1 atm H2at room temperature for 45 min. Then the resulting solution was filteredthrough 0.22 μm membrane, concentrated by lyophilization, and thenpurified with HPLC on a Develosil C30 RP Aqueous reverse phase column.The final product was obtained as a white solid (12 mg, 74%). HRMS(m/z): [M-H]-calculated for C₁₅H₂₄N₃O₁₆P₂, 564.0637; observed, 564.0634.

Synthesis of UDP-2-Keto-Glc (FIG. 2B; Compound 5).

UDP-2-Keto-Glc 5 was synthesized according to a published protocol[Dulcey, A. E., Qasba, P. K., Lamb, J. and Griffiths, G. L. (2011)Improved synthesis of UDP-2-(2-ketopropyl)galactose and a firstsynthesis of UDP-2-(2-ketopropyl)glucose for the site-specific linkingof biomolecules via modified glycan residues using glycosyltransferases.Tetrahedron, 67, 2013-2017], and its identity and purity were confirmedby HRMS, and analytical HPLC, respectively. HRMS (m/z): [M-H]-calculatedfor C₁₈H₂₇N₂O₁₇P₂, 605.0790; observed, 605.0776.

Example 3 Binding of Anti-Glucosamine (Anti-GlcN) Antibody to DNAModified with Glucosamine Derivatives

Antibody assays were performed with commercially availableanti-D-glucosamine antibody (Abcam, #ab62666). Results indicated thatanti-D-glucosamine antibody does not bind to C or 5-mC containing PCRT42K DNAs. For 5-hmC containing PCR T42K DNA treated with UDP-Glc,UDP-2-GlcN, UDP-6-GlcN, or UDP-6-Azido-Glc in the presence of BGT, only2-GlcN-containing DNA showed significant binding by theanti-D-glucosamine antibody. Anti-D-glucosamine antibody did not bind to5-hmC PCR T42K DNA containing 6-GlcN, 6-Azido-Glc or Glc.

FIG. 14 shows the results of the anti-D-glucosamine binding assay. Theassay was performed as follows: 400 ng of C, 5-mC, or 5-hmC PCR T42K DNAwas treated with BGT and UDP-2-glucosamine, UDP-6-glucosamine,UDP-6-Azido-Glc, or natural UDP-Glc, respectively, for 1 h at 37° C. andthen purified with QIA column. After measuring the O. D., DNA wasdiluted to proper concentration as desired. In this experiment,different amounts of DNA (for each part, first column: 150 ng, secondcolumn: 100 ng, third column: 50 ng) were denatured at 98° C. andspotted on a positively charged nylon membrane (Roche, #11209299001). UVcrosslinking (3 min on a transilluminator) or chemical crosslinking wasused to covalently attach the DNA to the membrane. The membrane wasblocked in 5% milk (1X PBS, 0.1% Tween) for 1 hour and thenanti-D-glucosamine antibody (Abcam, #ab62666) was added. The membranewas incubated at 4° C. for overnight and washed 3 times with 1X PBSTbuffer. The membrane was incubated with the secondary antibody,HRP-anti-rabbit IgG, for 1 hour, washed 3 times with 1X PBST buffer, andexposed to X-Film.

What is claimed is: 1.-26. (canceled)
 27. A composition comprising: aβ-glycosyltransferase; and UDP-glucosamine (UDP-GlcN).
 28. Thecomposition of claim 27, further comprising a buffer having a pH between6 and
 8. 29. The composition of claim 28, wherein the pH is between 6.8and 7.4.
 30. The composition of claim 27, further comprising arestriction enzyme capable of cleaving a nucleic acid at a sitecomprising β-glucosyl-5-hydroxymethylcytosine but not a site comprisingβ-2-glucosaminyl-5-hydroxymethylcytosine.
 31. A method for labeling amodified nucleotide in a nucleic acid, comprising: combining a nucleicacid having a modified nucleotide with the composition of claim 27 underconditions permitting the glucosamine to become covalently attached tothe modified nucleotide in the nucleic acid.
 32. A method according toclaim 31, further comprising reacting the covalently attachedglucosamine with a label.
 33. A method according to claim 31, whereinthe modified nucleotide is hydroxymethylated cytosine (hmC).
 34. Amethod according to claim 33, further comprising enriching, detecting,isolating and/or identifying the position of the glucosamine-attachednucleotide in the nucleic acid.
 35. A method according to claim 34,where the nucleic acid is DNA.
 36. A method for detecting a modifiednucleotide in a genome or genomic fragment, comprising: (a) combining anucleic acid having a modified nucleotide with UDP-glucosamine(UDP-GlcN) and T4-β-glucosyltransferase (BGT); (b) permitting theglucosamine to become covalently attached to the modified nucleotide inthe nucleic acid; and (c) detecting the modified nucleotide of (b) bymeans of a label or by modification-specific enzymatic cleavage.
 37. Amethod according to claim 36, wherein the enzyme is capable of cleaving5-hydroxymethylcytosine (5-hmC), β-glucosyl-5-hydroxymethylcytosine(5-gmC), but not glucosamine-containing 5-hmC or labeledglucosamine-containing 5-hmC sites.
 38. A method according to claim 36,wherein the enzyme is capable of cleaving cytosine, and/or5-methylcytosine (5-mC), and/or 5-hydroxymethylcytosine (5-hmC), but notglucosamine-containing 5-hmC or labeled glucosamine-containing 5-hmCsites.
 39. A method for detecting a modified nucleotide in a genome orgenomic fragment, comprising: (a) combining a nucleic acid having amodified nucleotide with UDP-glucosamine (UDP-GlcN) andT4-β-glucosyltransferase (BGT); (b) permitting the glucosamine to becomecovalently attached to the modified nucleotide in the nucleic acid; and(c) detecting the modified nucleotide of (b) by means of a nucleic acidmodification-specific polymerase reaction.
 40. A kit comprising aβ-glycosyltransferase and UDP-GlcN.
 41. A nucleic acid comprisingmodified β-glucosyl-5-hydroxymethylcytosine in which one or more of thehydroxyl groups at the 2- and/or 6-positions of the glucosyl moiety havebeen replaced with amino groups.
 42. The nucleic acid of claim 41,wherein the modified β-glucosyl-5-hydroxymethylcytosine is aβ-glucosaminyloxy-5-methylcytosine.
 43. The nucleic acid of claim 42,wherein the β-glucosaminyloxy-5-methylcytosine isβ-2-glucosaminyloxy-5-methylcytosine.
 44. The nucleic acid of claim 42,wherein the modified β-glucosyl-5-hydroxymethylcytosine isβ-6-glucosaminyloxy-5-methylcytosine.
 45. A nucleic acid according toclaim 41, further comprising a label.
 46. A mammalian genome or genomefragment comprising a nucleic acid according to claim
 41. 47. A methodfor labeling a modified nucleotide in a nucleic acid, comprisingreacting a nucleic acid according to claim 41 with a label at a modifiedβ-glucosyl-5-hydroxymethylcytosine.
 48. A method for detecting amodified nucleotide in a nucleic acid, comprising labeling a modifiednucleotide in a nucleic acid according to the method of claim 47, anddetecting the label.